Download Arsenic in Cancer Treatment: Challenges for Application of Realgar

Toxins 2010, 2, 1568-1581; doi:10.3390/toxins2061568
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toxins
ISSN 2072-6651
www.mdpi.com/journal/toxins
Review
Arsenic in Cancer Treatment: Challenges for Application of
Realgar Nanoparticles (A Minireview)
Peter Baláž 1 and Ján Sedlák 2,*
1
2
Institute of Geotechnics, Slovak Academy of Sciences, 043 53 Košice, Slovakia;
E-Mail: [email protected]
Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +421-2-5932-7260; Fax: +421-2-5932-7250.
Received: 18 April 2010; in revised form: 17 June 2010 / Accepted: 18 June 2010 /
Published: 21 June 2010
Abstract: While intensive efforts have been made for the treatment of cancer, this disease
is still the second leading cause of death in many countries. Metastatic breast cancer,
late-stage colon cancer, malignant melanoma, multiple myeloma, and other forms of cancer
are still essentially incurable in most cases. Recent advances in genomic technologies have
permitted the simultaneous evaluation of DNA sequence-based alterations together with
copy number gains and losses. The requirement for a multi-targeting approach is the
common theme that emerges from these studies. Therefore, the combination of new
targeted biological and cytotoxic agents is currently under investigation in multimodal
treatment regimens. Similarly, a combinational principle is applied in traditional Chinese
medicine, as formulas consist of several types of medicinal herbs or minerals, in which one
represents the principal component, and the others serve as adjuvant ones that assist the
effects, or facilitate the delivery, of the principal component. In Western medicine,
approximately 60 different arsenic preparations have been developed and used in
pharmacological history. In traditional Chinese medicines, different forms of mineral
arsenicals (orpiment—As2S3, realgar—As4S4, and arsenolite—arsenic trioxide, As2O3) are
used, and realgar alone is included in 22 oral remedies that are recognized by the Chinese
Pharmacopeia Committee (2005). It is known that a significant portion of some forms of
mineral arsenicals is poorly absorbed into the body, and would be unavailable to cause
systemic damage. This review primary focuses on the application of arsenic sulfide
(realgar) for treatment of various forms of cancer in vitro and in vivo.
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Keywords: arsenic sulfide; realgar; cancer; nanoparticle; milling
1. Introduction
While intensive efforts have been made for the treatment of cancer, this disease is still the second
leading cause of death in many countries. Nearly 10.9 million new cases were diagnosed in the year
2005. In spite of enormous effort, the mortality rate due to cancer did not change in the past five to six
decades [1]. Metastatic breast cancer, late-stage colon cancer, malignant melanoma, and other forms of
cancer are still essentially incurable in most cases. The past quarter century of outstanding progress in
fundamental cancer biology has not translated into even distantly comparable advances in the clinical
practice [2]. The effectiveness of cancer therapy is limited by the rapid removal of anticancer agents
from the circulation by metabolism or excretion, by the inability to access and penetrate the target
cancer cells, or by nonspecific uptake by sensitive normal cells and tissues [3].
A strategy could be to associate anticancer agents with nanoparticles so as to overcome mechanisms
of resistance and to increase the selectivity of such agents towards cancer cells, while reducing the
drugs’ toxicity towards normal cells and issues. If designed properly, nanoparticles (defined usually as
materials with a size < 100 nm) may act as drug vehicles that are able to target tumor tissues or cells,
to a certain extent, while protecting the agent from premature activation during its transport [4].
Because their properties differ from those of their bulk counterparts, nanoparticles offer a range of
potential applications in cancer treatment, especially in the targeting and imaging or cells or tissues.
This review primary focuses on the application of arsenic sulfide (As4S4, realgar, REA) for the
treatment of various forms of cancer in vitro and in vivo.
2. Medicinal Use of Arsenic Compounds
Several papers describing the effect of arsenic derived compounds for medicinal purposes have
been published recently [5–8].
2.1. History
Arsenic compounds had a long Janus-type interaction with humanity. On one hand, they have been
extensively utilized, but on the other hand, their poisonous properties have caused misery and many
deaths. Because of their significant properties, arsenic compounds have been used as therapeutic
agents for more than 2400 years [5]. In early times of the East, a mixture of orpiment, slaked lime
(CaO), and water was favored as a depilatory. Moreover, a paste of realgar was recommended by
Hippocrates for treatment of ulcers. Arsenic compounds have also been used to treat the plague,
malaria, and cancer. Arsenides and arsenic salts were key ingredients in antiseptics, antispasmodics,
antiperiodics, caustics, cholagogues, hematinics, sedatives, and tonics. Approximately 60 different
arsenic preparations have been developed and distributed during the history [7].
Among inorganic compounds of arsenic, Fowlers’ solution (potassium bicarbonate-based solution
of arsenic oxide As2O3) was used in the treatment of asthma, chorea, eczema, pemphigus, pernicious
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anemia, psoriasis, Hodgkin’s disease, and leukemia. However, in the early 20th century, this form of
cancer management was supplanted by radiation therapy and cytotoxic chemotherapy [8].
2.2. Arsenic oxide As2O3 (arsenolite, ATO) and tetraarsenic oxide As4O6 (TAO)
In Chinese traditional medicine, ATO has been applied for more than 500 years. Pishang, or white
arsenic, which essentially contained ATO, was used originally in the treatment of certain skin diseases
and asthma, and to promote the healing of surgical wounds [9]. Based on the principle in Chinese
traditional medicine of ―using a toxic agent against a toxic agent‖, in the early 1970s, a group of
clinical researchers at Harbin Medical University began to treat some types of cancer with ATO. These
initial treatments were followed by a careful clinical trial at the Shanghai Second Medical University,
which documented remarkable clinical efficacy in patients with newly diagnosed and relapsed acute
promyelocytic leukemia (APL) [8]. Patients were treated daily with 10 mg of intravenous ATO infused
over two to three hours. This monotherapy produced complete remission in six (85.7%) of seven patients
presenting with de novo APL [10]. The pivotal studies in China have been followed by investigations in
the United States. The induction of complete remission with low doses ATO in APL patients has been
confirmed [11]. The exhaustive list of clinical trials employing arsenic-based cancer drugs in
hematological malignancies and in solid tumors was published [6], while detailed overviews about the
signal transduction pathways and the transcription factors triggered by ATO in leukemic cells are
reviewed in [12]. It was suggested that alteration of cellular function and intracellular signal
transduction pathway modulations by arsenical drugs may result in the induction of apoptosis,
inhibition of growth and angiogenesis, and the promotion of differentiation [7]. Recently, the
proteolytic degradation of the oncoprotein, PML-RARA, as a general strategy for the eradication of
APL with ATO, due to the rapid clearing of leukemia initiating cells, was presented [13].
In 2003, novel orally administrable angiogenesis inhibitors were introduced by a Korean-American
team, and the ability of TAO to suppress several steps of the angiogenic process both in vitro and in
vivo was documented [14]. It was shown that this agent significantly decreased the proliferation,
migration, invasion, and tube formation of endothelial cells induced by the angiogenic factors. In
summary, TAO appears to be a novel antiangiogenic and antimetastatic chemical agent that can be orally
administered [14].
2.3. Arsenic sulfide As4S4 (realgar, REA) on scene
The use of arsenic sulfide As4S4 (realgar, REA) for battling cancer, in spite of a long tradition of
arsenic application for medicinal treatment, is seen only in a few countries. With the exception of
China—where application of REA can be traced for a very long time—in vitro experiments were
observed only in Korea [15], Singapore [16], and Slovakia [15,17].
2.4. Chinese traditional medicine
Realgar (Xiong Huang) is listed in the modern Chinese Pharmacopoeia and is suggested to be made
into pills or powders for use, and is taken at a dosage of 150–300 mg each time, with long-term
administration being avoided. The discovery that indigo (a purple dye obtained from certain plants)
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has a component, indirubin, which is clinically effective for leukemia, was made in the early 1960s,
when researchers at the Institute of Hematology, Chinese Academy of Sciences (Beijing) noted that an
herbal formula prescribed by Chinese doctors appeared to be producing good results in many APL
patients. A modified version of the traditional formula, which includes both indigo and REA with
Salvia miltiorrhiza, and tanshinone IIA as major active ingredients, was developed at this Institute [18].
The fundamental problem in REA application is its low solubility. REA in Niuhuang Jiedu Pian, a
common preparation for the common cold, has a low solubility in water, and only 4% is bioavailable
in physiological gastric juice or intestinal fluid [19]. The average total arsenic concentration in a
Niuhuang Jiedu Pian is approximately 7 ± 1% (i.e., 70,000 ppm), corresponding to 28 mg of arsenic
per pill; of this only 1 mg of arsenic finds its way into the blood stream, and 40% of this absorbed
arsenic (0.4 mg) is excreted in urine [19]. To overcome the low solubility and poor bioavailability,
realgar particles have been prepared and applied in nanosized dimensions (see coming text). The
average rate of DNA damage in the total body is represented by the urinary level of 8-OH-dG
(8-hydroxy-2′-deoxyguanosine), and is the most accepted biomarker of oxidative DNA damage. A
recent study [20] reveals that arsenite caused significantly higher urinary 8-OH-dG levels than both
realgar and orpiment on exposure to the same amount of compound. As the effectiveness of subsequent
DNA repair determines the outcomes of treatment, significant differences may exist between species, as
well as between individuals, due to inter-individual variation in DNA damage repair [21].
Both efficacy and side effects need to be considered in evaluating a drug. As described in previous
paragraphs, ATO is an effective drug in patients with APL, despite the occurrence of some severe side
effects. REA is almost insoluble in aqueous solution at neutral and slightly acidic conditions, and
consequently will exhibit much less toxicity and better tolerance, in comparison with patients treated
with ATO. NMR-based metabolomic analysis of urine, serum, and aqueous extracts of the liver tissue
of Wistar rats fed with REA highlighted a number of complex disturbances in the endogenous
metabolite profiles. Altered transmethylation, hypoglycemia, hyperlipoidemia, suspected reduction of
intestinal microflora, and oxidative injury of the liver are related to realgar induced biochemical
pathway perturbation [22]. It will be shown that REA is also effective for patients who relapsed after
treatment with ATRA or chemotherapy. Since smaller amounts of realgar are needed for the induction
of differentiation, than for apoptosis, the differentiation therapy deserves to be developed as a
complement to other current therapies [23].
2.5. Solid state properties
Realgar is a red compound that exists in several crystalline forms. At least two polymorphs of As4S4
(sometimes referred as As2S2 or AsS) have been reported to date. The room temperature stable α-As4S4
phase is structurally identical to mineral realgar [24]. A high-temperature phase, β-As4S4 can be
obtained by heating realgar above 260 °C. The β-phase can also be obtained by high-energy milling
[25,26]. The β-phase is stable and slowly reconverts to the α-phase upon cooling. Upon exposure to
visible light, both α- and β-phase transform from red materials into bright yellow pararealgar [27–30].
Both As4S4 phases consist of covalently bonded As4S4 cage-like molecules that have different crystal
structures. The molecules are held together by weak van der Waals interactions [29]. Recently, the
reaction mechanism underlying the photoinduced linkage isomerization of discrete As4S4 molecules in
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realgar to its pararealgar form was observed [31]. Because of the fact that arsenic occurs naturally in
many minerals and in groundwater in contact with geologic formations containing a high content of
arsenic, more than 20 prokaryotes utilize arsenic as a terminal electron acceptor for energy generation
that goes beyond the commonly used redox mechanisms of detoxification [32]. Among these
organisms, a distinct position is held by YeAs, which produces a biogenic mineral, -realgar [33].
Recently, the production of extracellular networks of filamentous arsenic-sulfide nanotubes by
Shewanella sp. strain HN-41 was discovered [34]. The formed nanotubes are initially amorphous
As2S3 that evolve with increasing time toward realgar and duranusite (AS4S). Upon maturation, the
arsenic sulfide nanotubes behaved as metals and semiconductors in terms of their electrical and
photoconductive properties, respectively.
2.6. Contemporary efforts
A pilot report from the Beijing University Institute of Hematology and the People’s Hospital group
dealt with the treatment of APL patients with REA that appeared in the blood [35]. Moderately
purified REA was mixed with an equal amount (w/w) of ground Semen platycladi, as an excipient, and
was put into capsules containing 250 mg of As4S4. This dosage was selected on the basis of a
dose-escalation trial and animal studies. In this small study, a group of volunteer patients showed that
escalation to a single dose of 60 mg/kg REA was well tolerated [36]. Hematologically complete
remission (CR) was achieved in all patients with newly diagnosed APL, as well as in those with
hematological relapse. Of 16 patients with newly diagnosed disease, and available cytogenetic and
molecular analysis, 14 had cytogenetic and molecular CR. Treatment with well tolerated REA had
only moderate side effects. Degeneration or apoptosis of APL promyelocytes was observed during
REA therapy. Most urinary arsenic excretion occurred within the first 24 hours, and both blood and
urine arsenic levels declined after discontinuation of REA. The results showed, for the first time, that
REA treatment alone is highly effective and safe in both remission induction and maintenance therapy
in patients with APL, regardless of disease stage [35]. Two years later, the first author of the described
work has been granted a patent in the United States [37].
2.7. Mechanism of REA effect
Treatment of promyelocytic NB4 or T-leukemia CEM cells with REA induced some of the
morphological features of apoptotic cells. The expression of APO 2.7, which is located at the
membrane of mitochondria, was upregulated [38]. An assessment of the sensitivity of NB4 and K562
cells to REA treatment revealed a low sensitivity of K562 cells, due to high expression of Bcl-x(L),
which provides resistance to REA treatment. The reversion of resistance is achieved by transfecting
bcl-x(L) antisense RNA vector; cells subsequently become sensitive to realgar at clinically acceptable
concentrations [39]. The expression profile analysis of NB4 cells treated with REA using 1K chip
revealed the upregulation of nine genes and the downregulation of 37 genes [40]. The most
pronounced effect of REA in NB4 cells—growth suppression and apoptosis—may by associated with
induced reorganization of PML oncogenic domains. These domains are involved in transcriptional
regulation and other different cellular functions [41]. The upregulation of proteasome PSMC2 and
PSMD1 proteins can contribute to the degradation of fusion protein PML/RARa and to the increase of
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Hsp90. These changes are consistent with stress-related cell responses [42], or with the facilitation of
interactions between abnormal protein and components of the proteasomal pathway. Examples of
genes with decreased expression are as follow: MERTK, which encodes for a c-mer tyrosine kinase
widely expressed in monocytes and macrophages, a regulator of G protein signaling 2 (RGS2) that
belongs to a group of G0/G1 switch regulatory genes, and which is expressed in leukemia cells, with
the exception of CML in the chronic phase and normal hematopoietic cells, genes that encode several
ribosomal proteins, protein-tyrosine phosphatase 4A, which, as a downstream effector of p53 is
involved in cell cycle regulation [43], and others.
In 2004, a study of the combined effects of REA and imatinib on CML cells and BCR-ABL
oncoprotein was published [44]. Analysis of cell proliferation and clonogenic ability showed that REA
and imatinib exerted synergistic effects on both K562 cells and on fresh CML cells. Both drugs under
study also exhibited a synergistic effect in targeting the BCR-ABL protein. While REA triggered its
degradation and imatinib inhibited its tyrosine kinase activity, their combined use led to lower
enzymatic activity levels of BCR-ABL. This combination represents a new model of synergistic
targeting at the molecular level and might be a promising approach for improving response rates and
for resistance prevention. The expression profile of human multiple myeloma RPMI8226 cells treated
with REA identifies the upregulation of 54 and the downregulation of 60 genes [45]. Upregulated
genes are associated with a variety of cell functions, such as the regulation of apoptosis, cell-cell
signaling, cell growth, and cell homeostasis. These genes include ones that code for the monocyte
chemotactic protein MCP1, the macrophage inflammatory protein MIP-1, several TNF-induced
proteins, and the binding protein for insulin-like growth factor IGFBP4, which is involved in the
systematic and local regulation of IGF activity, along with the B-cell translocation gene BTG1, which
negatively regulates cell proliferation. When comparing the extent of gene expression changes in
ATO-treated RPMI8226 cells, it was found that 121 genes were upregulated and 152 down-regulated,
most significantly thioredoxin-interacting protein TXNIP and activin receptor-like kinase ALK1 [46].
Results suggest that REA and ATO affect different set of genes. The additional differences in the
sensitivity of cells to REA treatment may originate from differences in the tissue phenotype that the
cells are derived from, because proliferation of normal human fibroblast Hs-68 cells was only slightly
influenced, while high cytotoxicity in HaCaT immortalized human epidermal keratinocytes was
observed [47].
CD11b expression and the NBT test confirmed that REA-induced differentiation of HL-60 cells is
preceded with marked changes in ROS and catalase activity, and with small changes in superoxide
dismutase activity and in levels of the reduced form of glutathione [48]. The effect of REA
nanoparticles on the differentiation of HL-60 cells was synergized, enhanced, and suppressed by the
inhibition of p38 MAPK, JNK, and ERK pathways, respectively [49]. The analysis of human
histocytic lymphoma U937 cell death, induced by REA powder, revealed that the time- and
concentration-dependent induction of apoptosis begins to decrease with an enhanced necrotic cell ratio
at high REA concentrations. The use of a panel of protein kinase inhibitors demonstrates the
involvement of the PI3-K/Akt signaling pathway in the downregulation of SIRT1 deacetylase activity,
the subsequent activation of p53, and apoptosis induction [50]. As SIRT1 deacetylates the DNA repair
factor Ku70, causing it to sequester the proapoptotic factor Bax away from mitochondria, SIRT1
inhibition can sensitize cells toward stress-induced apoptotic cell death [51]. Similar enhancement of
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the phosphoacetylation of the p53 paralog, p73, was observed in NB4 and K562 cells that were treated
with a combination of MEK inhibitor and ATO, which promotes a p300-mediated acetylation and the
p73-induced upregulation of p21 and Bax [52].
In paper [23], a trial has been described to compare the effect of REA and ATO on HL-60 cell
differentiation, a leukemic cell line that is blocked at the level of promyelocytic differentiation [53].
The differentiation is accompanied by changes in the expression of surface antigens and in the
acquisition of a number of functions indicative of cell differentiation into granulocyte-like or
monocyte or macrophage-like cells [54]. ATO has been reported to induce granulocytic differentiation
in HL-60 cells [55]. Although REA is similar to ATO in inducing HL-60 cell differentiation at
sub-micromolarity, the differentiation pathways are different. As shown in paper [56], the involvement
of Ser/Thr protein phosphatases in the differentiation induced by REA takes a place. The total
activities of PP1 (serine/threonine protein phosphatase type 1) and type 2A (PP 2A) are elevated with
increasing REA concentration (in the range of 0–2.0 μM), while the differentiation extent, as indicated
by the NBT reduction assay, peaked at 0.75 μM of REA. On the other hand, it was found that the
inhibitors of PP1, PP2a, and PP2B (cyclosporine A and FK 506) did not have any effect on
ATO-induced differentiation in HL-60 cells [57].
These results clearly indicate that realgar As4S4 is different from arsenic oxide As2O3 in the
differentiation pathway and molecular mechanism.
2.8. Nanomaterials for cancer treatment: realgar case
Modification of water insoluble drugs (like REA) into ultra-fine particles with large surface areas is
considered to be an efficient approach for improving bioavailability, and is practically important in the
pharmaceutical industry. The application of micronization, if applied in a proper way, can lead to mean
particle sizes of approximately 3–5 μm. However, many of the new drugs show such a low solubility
that even micronization does not lead to a sufficient increase in bioavailability after oral administration
[58]. Thus, by reducing particle sizes from micrometers to nanometers (nanonization), and thus,
increasing surface area, one or more order(s) of magnitude increase in dissolution rate can be achieved
[58–60]. Tumor vasculature is highly abnormal, proliferative, activated, and tortuous; it presents
increased permeability and gaps, with pores between 350 and 800 nm in diameter, and a cutoff around
400 nm [61]. Thus preparation of nanoparticles to be 100–200 nm in diameter by any nanosizing
technique gives a good predisposition for nanodrug penetration into a tumor body. While the reduction
of particle sizes has been employed in the pharmaceutical industry for several decades, recent advances
in milling technology have enabled the production of such nanoparticles in a reproducible manner.
Nanocrystalline dispersions are prepared using the media milling processes [62,63]. The media milling
process readily fractures micron-sized drug crystals into homogeneous nanoparticle dispersions.
There are a small amount of papers dealing with the application of nanorealgar (NREA) in cancer
treatment. In 2001, a paper has been published which deals with the size effects of realgar particles on
apoptosis in a human umbilical vein endothelial cell line ECV-304 [64]. Four different suspensions of
NREA particles, with diameters in the range of 100–500 nm, which contained equivalent doses, were
investigated to determine their size effects. Remarkable cytotoxic effects and induction of apoptosis
confirmed by cell morphology, flow cytometry analysis, and DNA ladder presence, was achieved by
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exposure of cells to realgar particles of diameters of 100 and 150 nm, whereas the treatment of cells
with particles of a diameter of 200 and 500 nm had only slight effects on cell viability. Authors
suggested that the inhibition of angiogenesis may account for the therapeutic effect of nanosized REA
particles. The concentration-dependent decrease of cancer cell line viability was observed, and the
most sensitive cells for nanosized REA are HL-60 and K562 cells, followed by the more resistant
multiple myeloma ARH 77 and U-266 cell lines [17].
In paper [65], the effect of long-term milling (six hours) of REA on induction of cytotoxicity in
human promyelocytic leukemia HL-60 cells has been studied. Such a milling mode usually brings
aggregation and agglomeration effects in solids [26]. As a post-milling procedure, authors prepared a
solid dispersion using polyethylene glycol (PEG 6000) as the carrier. Based on published SEM and
TEM images, one cannot be strongly convinced about the presence of nanoparticles, in spite of the
author’s statement of particle size (200 nm). The described milling performance needs a more
thorough, critical treatment because such a milling mode can bring contamination into realgar, as a
consequence of wear from the milling media. In spite of this drawback, the paper brings new insights.
It was found that treatment with NREA resulted in considerably lower cell viability compared with
that of raw realgar particles. On the other hand, treatment with NREA promoted the generation of
reactive oxygen species, inhibition of catalase activity, accompanied by lipid peroxidation and protein
oxidation, and the loss of protein free thiols, whereas such events were not observed in HL-60 cells
exposed to raw (higher diameter) REA particles. Another paper [66] shows that both NREA and raw
REA particles reduced membrane fluidity in HL-60 cells, while enhancements in LDH leakage and
membrane lipid peroxidation were observed in nanoparticle REA-treated cells, in comparison to
control or raw REA-treated cells. In addition, nanoparticle REA significantly decreased cell viability
and produced DNA ladder formation, along with the appearance of cells at the sub-G1 phase, whereas
raw REA particles failed to produce characteristic apoptotic events. Treatment of HL-60 cells with
REA powder filtered through a 0.22 m filter membrane revealed that membrane lipid peroxidation
was achieved at the lowest concentration of REA and LDH leakage, and DNA fragmentation occurred
at higher concentrations used. Analysis of atomic force microscope images confirmed the existence of
a disorganized membrane surface consisting of protruding protein clusters and confirmed the
membrane toxic effect of REA in HL-60 cells [67].
The technique of cryo-milling has been applied for the first time at the National University of
Singapore for the preparation of NREA samples to evaluate their in vitro activity and in vivo
bioavailability [16]. The particle size of NREA ground in the presence of polyvinylpyrrolidone (PVP)
or sodium dodecyl sulphate (SDS) was in the range from 176 to 243 nm, and was measured by means
of a Zetasizer, while TEM images revealed a size of about 50 nm. The plausible explanation is that the
particles observed by TEM were the individual realgar particles, whilst the particles apparent to the
Zetasizer were the realgar particles with PVP or SDS coating(s), or both. The cytotoxic and
anti-proliferative effects of such NREA particles in human ovarian CI80-13S, OVCAR, OVCAR-3,
and cervical HeLa cancer cell lines were observed. CI80-13S are most sensitive to the NREA with IC50
values of less than 1 μM, whereas the other cancer cell lines had IC50 values in a range of 2–4 μM
NREA. The cytotoxic activity of the NREA particles to these human gynecological cell lines was
comparable to ATO cytotoxic effects and was due to apoptosis induction. Bioavailability
investigations revealed a remarkable increase in the urinary recovery of arsenic in rats after a single
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oral administration of the NREA particle suspension. The administered dose of arsenic recovered in
urine from the NREA preparation in presence of PVP or SDS, or both, ranged from 59–79%, whereas
the original REA powder gave a urinary recovery of only 25%. The improvement in bioavailability can
be attributed to the realgar particles’ size reduction.
It is well known that cysteine-dependent aspartame specific proteases (caspases) are an important
mediator of signal transduction in apoptosis. Both induction of apoptosis and prevention of
angiogenesis enhance the anticancer effect of the transferal delivery of REA nanoparticles against
mouse melanoma B16 cells. In this treatment, there seems to be no apparent in vivo toxic
manifestation, as evaluated in terms of body weight changes, feeding behavior, skin irritation, and liver
injury [68]. REA nanoparticle-induced apoptosis in U937 cells was partially prevented by inhibitors of
caspase-3, -8, and -9 [69]. While inhibitors of ERK and p38 kinases failed to block cell death, the
inhibitor of JNK significantly inhibits REA-induced apoptosis. The REA-induced increase of the
Bax/Bcl-2 ratio and the phosphorylation of JNK are involved in U937 apoptosis. Similarly, the
proliferation and viability of CML cell line K562 is affected by micromolar concentrations of REA.
The REA treatment decreases Bcr-Abl protein content, although the mRNA level of the bcr-abl fusion
gene does not change. Consequently the PTK activity of c-Abl and Bcr-Abl was decreased in a timeand concentration-dependent manner. Compared with CML Ph+ leukemia cells, Ph− mononuclear
cells were less sensitive to REA treatment at all concentrations tested. When targeting the Bcr-Abl, the
crucial factor of CML pathogenesis and the higher sensitivity of Ph+ cells to apoptosis induction
suggests the therapeutic potential of REA for CML patients [70].
3. Conclusion
In traditional Chinese medicine, there is well established multicomponent characters for remedies.
The combination of arsenicals with natural compounds can modify unpleasant side effects, such as
cardiotoxicity or genotoxicity in normal cells [71,72] or enhance the cytotoxic effect [18]. On the other
hand, the effective surface modification of insoluble arsenic particles with ligand targeted to tumor
cells represents a promising tool for the enhanced hit-and-kill antitumor effect of future nanomedicine
approaches to cancer patient treatment.
Acknowledgement
This work was partially supported by grants from The Slovak Research and Development Agency
under the contracts VVCE-0001-07 and LPP-0107-09, and Center of Excellence of Slovak Academy
of Sciences NANOSMART.
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